Botulism was first described almost 200 years ago. This disease is caused by the botulinum neurotoxins (BoNT), which are the most potent toxins known. There are seven related toxins (also called serotypes), A-G, produced mainly by toxigenic strains of Clostridium botulinum. Each toxin is composed of a heavy (H) chain and a light (L) chain;the H-chain mediates binding of the toxin to specific receptors that are exposed (at least transiently) on the surface of neurons. Upon endocytosis of toxin-receptor complexes, acidification of the lumen of the internalized vesicle triggers the H-chain to form a putative channel-like structure that serves to translocate the L-chain in the cytosol. The L-chain is a zinc dependent protease. Following translocation, the L- chain specifically cleaves SNARE proteins in the cytosol, thereby blocking the release of neurotransmitters at the neuromuscular junction. This can result in paralysis and death. Most of the BoNTs are thought to bind to the surface of neurons via a dual-receptor mechanism in which the receptor consists of two components: gangliosides and protein(s). We previously identified protein receptors for four BoNTs: A, B, E and G. In Specific Aim 1 we seek to continue these studies by focusing on the entry pathway and protein receptor for BoNT/F. BoNT/F is the only remaining serotype - known to cause disease in humans (along with A, B and E) - for which the receptor is unknown. Our new preliminary data indicate that BoNT/F enters via recycling synaptic vesicles, but utilizes a novel receptor that is not involved in the entry of the other BoNTs for which protein receptors have been identified. In this Aim we will also address the role of gangliosides as putative co- receptors for BoNT/F and we will engineer chimeric receptors that mediate entry of BoNTs into a wide range of cell types, thus expanding their potential medical applications. In Specific Aim 2 we will explore the least understood step in the action of the BoNTs: H-chain mediated translocation of the L-chain across membranes. A combination of biochemical, biophysical and cell-based experiments will be carried out to determine the order of assembly, and structure, of the potentially oligomeric membrane-bound form of the H-chain. These studies will emphasize the influence of receptors on the ability of the H-chain to sense pH and to assemble into 'translocation machines'. Finally, in Specific Aim 3, we will directly address an emerging hypothesis: following injection into the periphery (i.e. at the neuromuscular junction) to treat disease, BoNT/A - and perhaps other toxin serotypes - are potentially transported into the central nervous system to cause some of their effects. We have devised a simple model system, using cultured neurons and toxins labeled with quantum dots, to study the putative movement of BoNTs within and between neurons. This system is amenable to molecular analysis, making it possible to directly visualize toxin transport and transcytosis and to elucidate the underlying mechanisms. These studies will directly address the question, and means, by which BoNTs affect the central nervous system following peripheral injection.